Eukaryotic elongation factor 2 protein kinase (eEF2K) is a ubiquitously expressed protein that belongs to a family of alpha kinases characterized by an atypical kinase domain. Our recent findings demonstrate that inhibition or genetic inactivation of eEF2K protects normal tissues from cytotoxic effects of ionizing radiation and chemotherapeutic agents. We have previously generated eEF2K-deficient animals that have no adverse phenotypes but demonstrate increased resistance to both lethal doses of radiation and doxorubicin-induced cytotoxicity. This radio- and chemoresistant phenotype is accompanied by decreased levels of apoptosis in proliferating tissues. Moreover, siRNA-mediated knockdown of eEF2K sensitizes cancer cells to nutrient deprivation and to treatment with chemotherapeutic agent doxorubicin and suppresses tumor growth in the mouse model of breast cancer. Thus, we expect eEF2K inhibitors to exert dual effects in protecting normal tissues while enhancing tumor killing during chemotherapy, thereby significantly enhancing the therapeutic index of conventional chemotherapy. Several attempts have been made to develop chemical inhibitors of eEF2K, but the resulting compounds suffered from various limitations, such as low specificity for eEF2K, insufficient potency, high serum binding, or high toxicity, precluding their application in the clinic. We have developed a novel screening platform enabling rapid and accurate determination of eEF2K phosphorylation activity in vitro. Using this platform, we have generated a series of new eEF2K inhibitors that can be produced with high yields and purity and exhibit high specificity, improved potency, low serum binding, and low toxicity. Our preliminary data show that this series of eEF2K inhibitors effectively mimics the effects of siRNA-mediated knockdown of eEF2K expression, sensitizing cancer cells to nutrient deprivation and doxorubicin. Moreover, our lead inhibitor compound reduced doxorubicin toxicity in vivo, as evidenced by a reduction of a biomarker of tissue damage. In this grant we propose to further optimize our eEF2K inhibitor series and to carry out a panel of proof-of-concept experiments testing the efficacy of these inhibitors in sensitizing cancer cells to nutrient deprivation and doxorubicin treatment and suppressing tumor growth in a mouse breast cancer model. It is anticipated that upon completion of this project we will identify an inhibitor of eEF2K that will potentiate tumor killing by chemotherapy while protecting normal tissues from the associated toxicity, thereby representing an unprecedented approach to cancer therapy.